Correct determination of the instantaneous level and changes of relevant proteins inside individual cells is essential for correct interpretation and understanding of physiological, diagnostic, and therapeutic events. Thus, single-cell analyses are important for quantification of natural cellular heterogeneity, which cannot be evaluated from averaged data of a cell population measurements. Here, we developed an original highly sensitive and selective instrumentation and methodology based on homogeneous single-step bioluminescence assay to quantify caspases and evaluate their heterogeneity in individual cells. Individual suspended cells are selected under microscope and reliably transferred into the 7 μl detection vials by a micromanipulator. The sensitivity of the method is given by implementation of photomultiplying tube with a cooled photocathode working in the photon counting mode. By optimization of our device and methodology, the limits of detection and quantitation were decreased down to 2.1 and 7.0 fg of recombinant caspase-3, respectively. These masses are lower than average amounts of caspase-3/7 in individual apoptotic and even non-apoptotic cells. As a proof of concept, the content of caspase-3/7 in single treated and untreated HeLa cells was determined to be 154 and 25 fg, respectively. Based on these results, we aim to use the technology for investigations of non-apoptotic functions of caspases.

• MeSH
• Apoptosis * MeSH
• HeLa Cells MeSH
• Caspase 3 MeSH
• Caspases * MeSH
• Humans MeSH
• Technology MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Caspase-2 is an apical protease responsible for the proteolysis of cellular substrates directly involved in mediating apoptotic signaling cascades. Caspase-2 activation is inhibited by phosphorylation followed by binding to the scaffolding protein 14-3-3, which recognizes two phosphoserines located in the linker between the caspase recruitment domain and the p19 domains of the caspase-2 zymogen. However, the structural details of this interaction and the exact role of 14-3-3 in the regulation of caspase-2 activation remain unclear. Moreover, the caspase-2 region with both 14-3-3-binding motifs also contains the nuclear localization sequence (NLS), thus suggesting that 14-3-3 binding may regulate the subcellular localization of caspase-2. Here, we report a structural analysis of the 14-3-3ζ:caspase-2 complex using a combined approach based on small angle X-ray scattering, NMR, chemical cross-linking, and fluorescence spectroscopy. The structural model proposed in this study suggests that phosphorylated caspase-2 and 14-3-3ζ form a compact and rigid complex in which the p19 and the p12 domains of caspase-2 are positioned within the central channel of the 14-3-3 dimer and stabilized through interactions with the C-terminal helices of both 14-3-3ζ protomers. In this conformation, the surface of the p12 domain, which is involved in caspase-2 activation by dimerization, is sterically occluded by the 14-3-3 dimer, thereby likely preventing caspase-2 activation. In addition, 14-3-3 protein binding to caspase-2 masks its NLS. Therefore, our results suggest that 14-3-3 protein binding to caspase-2 may play a key role in regulating caspase-2 activation. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.ww pdb.org (PDB ID codes 6GKF and 6GKG).

• MeSH
• Cysteine Endopeptidases chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Nuclear Localization Signals * MeSH
• Caspase 2 chemistry   metabolism   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Scattering, Small Angle MeSH
• Models, Molecular MeSH
• 14-3-3 Proteins chemistry   metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Among all species, caspase-2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164 , within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14-3-3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14-3-3-dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14-3-3 by hydrogen/deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14-3-3-mediated inhibition of C2 activation. Our data reveal that the 14-3-3 dimer interacts with proC2 not only through ligand-binding grooves but also through other regions outside the central channel, thus explaining the isoform-dependent specificity of 14-3-3 protein binding to proC2 and the substantially higher binding affinity of 14-3-3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14-3-3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14-3-3 protein interacts with and masks both the nuclear localization sequence and the C-terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14-3-3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14-3-3 protein likely inhibits proC2 activation by blocking its dimerization surface. DATABASES: Structural data are available in the Protein Data Bank under the accession numbers 6SAD and 6S9K.

• MeSH
• Phosphorylation MeSH
• Caspase 2 chemistry   genetics   metabolism   MeSH
• Protein Conformation * MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular * MeSH
• Protein Multimerization * MeSH
• Mutation MeSH
• Protein Isoforms genetics   metabolism   MeSH
• Protein Precursors chemistry   genetics   metabolism   MeSH
• 14-3-3 Proteins chemistry   genetics   metabolism   MeSH
• Recombinant Proteins chemistry   metabolism   MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Caspase-3 is an executive caspase, in the central position within apoptotic machinery. Apoptosis as a way of programmed cell death is a physiological process that plays an essential role in the development and homeostasis maintenance; moreover, its deregulations are linked to tumor progression or various autoimmune disorders. Therefore, an investigation of apoptosis pathways on the level of individual cells is not only of biological but also medical importance. In this work we report on the development of a high-sensitivity instrumentation and protocol for detection of active caspase-3 in individual mammalian apoptotic cells. The technology is based on the specific cleavage of modified luciferin by caspase-3, an immediate bioluminescence reaction of free luciferin with luciferase followed by emissions of photons and their detection by photomultiplier tube working in the photon counting regime. Three different instrumental arrangements are compared for the determination of caspase-3 in free cells or tissue samples. Thus, in our best miniaturized system the mean amount as low as about 6.5 fg corresponding to 122 000 molecules of caspase-3 can be detected in individual apoptotic mouse leg cells.

• MeSH
• Single-Cell Analysis instrumentation   methods   MeSH
• Apoptosis physiology   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Caspase 3 analysis   metabolism   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Luminescent Measurements instrumentation   methods   MeSH
• Mice MeSH
• Pregnancy MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Pregnancy MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Tooth morphogenesis is accompanied by apoptotic events which show restricted temporospatial patterns suggesting multiple roles in odontogenesis. Dental apoptosis seems to be caspase dependent and caspase-3 has been shown to be activated during dental apoptosis.Caspase-3 mutant mice on different genetic backgrounds were used to investigate alterations in dental apoptosis and molar tooth morphogenesis. Mouse embryos at E15.5 were analyzed to reveal any changes in enamel knots, which are transient structures eliminated by apoptosis. In caspase-3(-/-) mice on the B57BL/6 background, disorganization of the epithelium was found in the original primary enamel knot area and confirmed by altered expression of Shh. Despite this early defect in molar tooth development, these mutants showed correct formation of secondary enamel knots as indicated by Fgf-4 expression. Analyses of adult molar teeth did not reveal any major alterations in tooth shape, enamel structure or pattern when compared to heterozygote littermates. In caspase-3(-/-) mice on the 129X1/SvJ background, no defects in tooth development were found except the position of the upper molars which developed more posteriorly in the oral cavity. This is likely, however, to be a secondary defect caused by a physical squashing of the face by the malformed brain. The results suggest that although caspase-3 becomes activated and may be essential for dental apoptosis, it does not seem fundamental for formation of normal mineralised molar teeth.

• MeSH
• Apoptosis MeSH
• Fibroblast Growth Factor 4 genetics   MeSH
• Financing, Organized MeSH
• In Situ Hybridization MeSH
• Caspase 3 MeSH
• Caspases genetics   deficiency   MeSH
• RNA, Messenger metabolism   MeSH
• Molar cytology   embryology   enzymology   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Odontogenesis physiology   genetics   MeSH
• Cell Proliferation MeSH
• Hedgehog Proteins MeSH
• Trans-Activators genetics   MeSH
• Gene Expression Regulation, Developmental MeSH
• Dental Enamel cytology   embryology   enzymology   MeSH
• Tooth Germ cytology   embryology   enzymology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH

Caspases are key enzymatic components of the intracellular apoptotic machinery, and their role in mammalian systems is often studied using fluoromethylketone (FMK) inhibitors. Despite many advantages of such approach, efficiency of the inhibitor and membrane permeability speed are often questioned. This work therefore focuses on an exact evaluation of caspase-3 FMK inhibition dynamics in camptothecin-induced mesenchymal micromasses. Two parameters of caspase-3 FMK inhibitor were investigated: first, the stability of the inhibitory potential in the time course of cultivation and, simultaneously, the dynamics of caspase-3 FMK inhibition after camptothecin-induced apoptosis peak. A photon-counting chemiluminescence approach was applied for quantification of active caspase-3. The sensitivity of the photon-counting method allowed for evaluation of active caspase-3 concentration in femtogram amounts per cell. The inhibitor penetrated the cells within the first minute after its application, and the peak of caspase-3 started to decline to the blank level after 30 min. The inhibitory effect of the FMK inhibitor was unchanged during the entire 48 h of cultivation.

• MeSH
• Apoptosis drug effects   MeSH
• Mice, Inbred Strains MeSH
• Caspase Inhibitors pharmacology   MeSH
• Camptothecin pharmacology   MeSH
• Caspase 3 metabolism   physiology   MeSH
• Cells, Cultured MeSH
• Luminescent Measurements methods   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Fluorochrome-labeled inhibitors of caspases (FLICA) have been designed as an alternative tool for the detection of caspase activation in whole cells. They should label the active site of the corresponding caspase through a covalent attachment to the reactive cysteine residue. METHODS: One of the FLICAs, FAM-DEVD-fmk, was used to monitor apoptosis progression in leukemic JURL-MK1 cells by means of flow cytometry. The effects of unlabeled caspase inhibitors z-DEVD-fmk and z-VAD-fmk on FLICA staining were analyzed to evaluate the contribution of caspase-bound FLICA to the fluorescent signal. Covalent binding of inhibitors to caspase-3 subunit was revealed by Western blotting. RESULTS: Although the unlabeled inhibitors irreversibly bind to caspase-3, completely inhibit its activity, and prevent FLICA binding to caspase-3 even at concentrations lower than 5 muM, they have no effect on FLICA staining of apoptotic cells. CONCLUSIONS: Fluorescent signal of FLICA is characteristic for apoptotic cells but originates mainly from yet unspecified site(s) that differ from the caspase active site. This finding puts in doubt the specificity of staining by various FLICAs with regard to individual caspases and shows the need for an extreme care in the interpretation of data obtained using these labels. Copyright 2007 International Society for Analytical Cytology.

• MeSH
• Apoptosis MeSH
• Staining and Labeling MeSH
• Amino Acid Chloromethyl Ketones analysis   pharmacology   metabolism   MeSH
• Financing, Organized MeSH
• Fluorescent Dyes analysis   metabolism   MeSH
• Caspase Inhibitors MeSH
• Caspase 3 MeSH
• Kinetics MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Protein Subunits metabolism   MeSH
• Protein Processing, Post-Translational drug effects   MeSH
• Propidium MeSH
• Flow Cytometry MeSH
• Reproducibility of Results MeSH
• Sensitivity and Specificity MeSH
• Substrate Specificity MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH

Analysing the chemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo®3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.

• MeSH
• Single-Cell Analysis instrumentation   methods   MeSH
• Apoptosis * MeSH
• Equipment Design MeSH
• Enzyme Assays instrumentation   methods   MeSH
• Caspase 3 analysis   metabolism   MeSH
• Caspase 7 analysis   metabolism   MeSH
• Cells, Cultured MeSH
• Luminescent Measurements instrumentation   methods   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

We recently demonstrated that TLCK and TPCK could act as potent but nonspecific inhibitors of mature caspases [Frydrych and Mlejnek [2008] J Cell Biochem 103:1646-1656]. The question whether TLCK and TPCK inhibit simultaneously caspase activation and/or processing remained, however, open. In this article, we demonstrated that TPCK even enhanced caspase-3 and caspase-7 processing although it substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity in HL-60 cells exposed to various cell death inducing stimuli. Under the same conditions, TLCK had no effect or affected caspase-3 and caspase-7 processing marginally depending on cell treatment used. Importantly, TLCK substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity irrespectively to the treatment used. Interestingly, treatment of cells with toxic concentrations of TPCK alone was accompanied by full caspase-3 and -7 processing even if it induced necrosis. In contrast, treatment of cells with concentrations of TLCK that caused necrosis was accompanied by only partial caspase-3 and caspase-7 processing. Our results clearly indicated that TPCK and TLCK did not inhibit caspase-3 and -7 enzymatic activity by prevention of their activation and/or processing.

• MeSH
• Apoptosis MeSH
• HL-60 Cells MeSH
• Caspase Inhibitors MeSH
• Serine Proteinase Inhibitors pharmacology   MeSH
• Caspase 3 metabolism   MeSH
• Caspase 7 metabolism   MeSH
• Humans MeSH
• Tosylphenylalanyl Chloromethyl Ketone pharmacology   MeSH
• Tosyllysine Chloromethyl Ketone pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

Caspases are key enzymes activated during the apoptotic machinery. Apoptosis as a way of programmed cell death becomes deregulated in some pathologies including cancer transformations, neurodegenerative, or autoimmune diseases. Most of the methods available for the detection of apoptosis and caspases provide qualitative information only or quantification data as an average from cell populations or cell lysates. Several reports point to the importance of more accurate single-cell analyses in biomedical studies due to heterogeneity at tissue as well as cell level. To meet these requirements, we developed a miniaturized device enabling detection and quantification of active caspase-3/7 in individual cells at a femtogram level (10(-15) g). The active caspase-3/7 detection protocol is based on the bioluminescence chemistry commercially available as a Caspase-Glo™ 3/7 reagent developed by Promega. As a model, we used human stem cells treated by camptothecin to induce apoptosis. Individual apoptotic cells were captured from a culture medium under a microscope and transferred by a micromanipulation system into a detection capillary containing 2 μl of the reagent. Cells without activation by camptothecin served as negative controls. The detection limit of active caspase-3/7 achieved in the miniaturized system was determined as 0.20 and limit of quantification as 0.65 of the amount found in a single apoptotic human stem cell. Such a sensitive method could have a wide application potential in laboratory medicine and related clinically oriented research.

• MeSH
• Single-Cell Analysis instrumentation   MeSH
• Apoptosis * MeSH
• Cell Differentiation MeSH
• Neural Crest cytology   MeSH
• Equipment Design MeSH
• Camptothecin chemistry   MeSH
• Caspase 3 metabolism   MeSH
• Caspase 7 metabolism   MeSH
• Stem Cells drug effects   pathology   MeSH
• Humans MeSH
• Luminescence MeSH
• Micromanipulation MeSH
• Miniaturization instrumentation   MeSH
• Reproducibility of Results MeSH
• Inflammation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH